Exposure to environmental hazards induces oxidative stress and promotes deleterious modifications to the structure of DNA. These modifications are potentially mutagenic and can promote numerous human maladies, including cancer. The base excision repair (BER) pathway is the cells primary defense against oxidative DNA damage and maintains genome stability. To this point, genetic polymorphisms and defects in key BER enzymes show up in several human populations, and are often associated with an increased cancer risk. An essential BER enzyme is human apurinic/apyrimidinic (AP) endonuclease 1 (APE1), which is a multifunctional enzyme that processes DNA damage during BER. Utilizing the same active site, APE1 performs both AP endonuclease (endo) and 3' to 5' exonuclease (exo) activities. APE1 endo activity has been rigorously characterized. In contrast, the mechanism for APE1 exo activity remains elusive, and it is unclear how the compact active site can accommodate both an endo substrate (abasic site) and an exo substrate (3' mismatched or damaged base). Moreover, the channeling of toxic DNA intermediates by the BER co-complex during APE1 exo activities remains entirely unstudied, leaving a significant gap in our understanding of BER. Therefore, the objective of this proposal is to determine the APE1 exo mechanism during repair of mismatched and damaged DNA ends. We will place this activity in context of the larger DNA repair co-complex during BER substrate channeling. We hypothesize the exo reaction of APE1 is dependent on unique active site contacts to open the binding pocket during proofreading and the processing of damaged DNA ends. We additionally predict exo substrates promote DNA substrate channeling between APE1 and DNA polymerase beta (the next enzyme in the pathway) during BER. To test this, we propose the following aims: (1) Determine the mechanism of APE1 exo activity during BER proofreading; (2) Determine the mechanism of APE1 catalyzed removal of 3?-PG end damage; and (3) Determine the mechanism of BER substrate channeling during APE1 exo activity. To accomplish these aims we will utilize time-lapse X-ray crystallography to observe catalysis at the atomic level, and pre-steady-state enzyme kinetics to parse out the rates of important steps during catalysis. To address the mechanism of substrate channeling during APE1 exo activity, we will use single-molecule total internal reflection microscopy (TIRFM) to observe the assembly/disassembly of BER complexes on DNA. Small angle neutron scattering will complement the TIRFM studies by determining a structural envelope of the BER co-complex. Using this multidisciplinary approach, we will cast light on previously understudied APE1 DNA repair mechanisms. With this information in hand, we will be closer to our long-term goal of providing a basis for rational drug design towards the development of more effective chemotherapeutics and synergistic drug combinations that target proteins involved in the DNA damage response. This approach has proven successful for proteins central to DNA repair pathways, such as PARP-1.